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1.
J Clin Microbiol ; 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-32229603

RESUMO

Amplicon sequencing of 16S rRNA gene is commonly used for the identification of bacterial isolates in diagnostic laboratories, and mostly relies on the Sanger sequencing method. The latter, however, suffers from a number of limitations with the most significant being the inability to resolve mixed amplicons when closely related species are co-amplified from a mixed culture. This often leads to either increased turnaround time or absence of usable sequence data. Short-read NGS technologies could solve the mixed amplicon issue, but would lack both cost efficiency at low throughput and fast turnaround times. Nanopore sequencing developed by Oxford Nanopore Technologies (ONT) could solve those issues by enabling flexible number of samples per run and adjustable sequencing time. Here we report on the development of a standardized laboratory workflow combined with a fully automated analysis pipeline LORCAN (Long Read Consensus ANalysis), which together provide a sample-to-report solution for amplicon sequencing and taxonomic identification of the resulting consensus sequences. Validation of the approach was conducted on a panel of reference strains and on clinical samples consisting of single or mixed rRNA amplicons associated with various bacterial genera by direct comparison to the corresponding Sanger sequences. Additionally, simulated read and amplicon mixtures were used to assess LORCAN's behaviour when dealing with samples with known cross-contamination level. We demonstrate that by combining ONT amplicon sequencing results with LORCAN, the accuracy of Sanger sequencing can be closely matched (>99.6% sequence identity) and that mixed samples can be resolved at the single base resolution level. The presented approach has the potential to significantly improve the flexibility, reliability and availability of amplicon sequencing in diagnostic settings.

2.
Genes (Basel) ; 10(9)2019 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-31466373

RESUMO

Shotgun metagenomics using next generation sequencing (NGS) is a promising technique to analyze both DNA and RNA microbial material from patient samples. Mostly used in a research setting, it is now increasingly being used in the clinical realm as well, notably to support diagnosis of viral infections, thereby calling for quality control and the implementation of ring trials (RT) to benchmark pipelines and ensure comparable results. The Swiss NGS clinical virology community therefore decided to conduct a RT in 2018, in order to benchmark current metagenomic workflows used at Swiss clinical virology laboratories, and thereby contribute to the definition of common best practices. The RT consisted of two parts (increments), in order to disentangle the variability arising from the experimental compared to the bioinformatics parts of the laboratory pipeline. In addition, the RT was also designed to assess the impact of databases compared to bioinformatics algorithms on the final results, by asking participants to perform the bioinformatics analysis with a common database, in addition to using their own in-house database. Five laboratories participated in the RT (seven pipelines were tested). We observed that the algorithms had a stronger impact on the overall performance than the choice of the reference database. Our results also suggest that differences in sample preparation can lead to significant differences in the performance, and that laboratories should aim for at least 5-10 Mio reads per sample and use depth of coverage in addition to other interpretation metrics such as the percent of coverage. Performance was generally lower when increasing the number of viruses per sample. The lessons learned from this pilot study will be useful for the development of larger-scale RTs to serve as regular quality control tests for laboratories performing NGS analyses of viruses in a clinical setting.


Assuntos
Serviços de Laboratório Clínico/normas , Genoma Viral , Ensaio de Proficiência Laboratorial/métodos , Metagenoma , Metagenômica/normas , Análise de Sequência/normas , Genoma Humano , Humanos , Metagenômica/métodos , Análise de Sequência/métodos , Suíça
3.
ISME J ; 13(11): 2764-2777, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31292537

RESUMO

The most abundant aquatic microbes are small in cell and genome size. Genome-streamlining theory predicts gene loss caused by evolutionary selection driven by environmental factors, favouring superior competitors for limiting resources. However, evolutionary histories of such abundant, genome-streamlined microbes remain largely unknown. Here we reconstruct the series of steps in the evolution of some of the most abundant genome-streamlined microbes in freshwaters ("Ca. Methylopumilus") and oceans (marine lineage OM43). A broad genomic spectrum is visible in the family Methylophilaceae (Betaproteobacteria), from sediment microbes with medium-sized genomes (2-3 Mbp genome size), an occasionally blooming pelagic intermediate (1.7 Mbp), and the most reduced pelagic forms (1.3 Mbp). We show that a habitat transition from freshwater sediment to the relatively oligotrophic pelagial was accompanied by progressive gene loss and adaptive gains. Gene loss has mainly affected functions not necessarily required or advantageous in the pelagial or is encoded by redundant pathways. Likewise, we identified genes providing adaptations to oligotrophic conditions that have been transmitted horizontally from pelagic freshwater microbes. Remarkably, the secondary transition from the pelagial of lakes to the oceans required only slight modifications, i.e., adaptations to higher salinity, gained via horizontal gene transfer from indigenous microbes. Our study provides first genomic evidence of genome reduction taking place during habitat transitions. In this regard, the family Methylophilaceae is an exceptional model for tracing the evolutionary history of genome streamlining as such a collection of evolutionarily related microbes from different habitats is rare in the microbial world.

4.
BMC Res Notes ; 11(1): 135, 2018 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-29454387

RESUMO

OBJECTIVE: Lauric acid (C12) is a medium-chain fatty acid that inhibits growth and production of the greenhouse gas methane by rumen methanogens such as Methanobrevibacter ruminantium. To understand the inhibitory mechanism of C12, a transcriptome analysis was performed in M. ruminantium strain M1 (DSM 1093) using RNA-Seq. RESULTS: Pure cell cultures in the exponential growth phase were treated with 0.4 mg/ml C12, dissolved in dimethyl sulfoxide (DMSO), for 1 h and transcriptomic changes were compared to DMSO-only treated cells (final DMSO concentration 0.2%). Exposure to C12 resulted in differential expression of 163 of the 2280 genes in the M1 genome (maximum log2-fold change 6.6). Remarkably, C12 hardly affected the expression of genes involved in methanogenesis. Instead, most affected genes encode cell-surface associated proteins (adhesion-like proteins, membrane-associated transporters and hydrogenases), and proteins involved in detoxification or DNA-repair processes. Enrichment analysis on the genes regulated in the C12-treated group showed a significant enrichment for categories 'cell surface' and 'mobile elements' (activated by C12), and for the categories 'regulation' and 'protein fate' (represssed). These results are useful to generate and test specific hypotheses on the mechanism how C12 affects rumen methanogens.


Assuntos
Ácidos Láuricos/farmacologia , Metano/metabolismo , Methanobrevibacter/efeitos dos fármacos , Rúmen/microbiologia , Transcriptoma/efeitos dos fármacos , Animais , Técnicas de Cultura de Células , Methanobrevibacter/genética , Análise de Sequência de RNA
5.
ISME J ; 12(1): 185-198, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29027997

RESUMO

Actinobacteria of the acI lineage are the most abundant microbes in freshwater systems, but there are so far no pure living cultures of these organisms, possibly because of metabolic dependencies on other microbes. This, in turn, has hampered an in-depth assessment of the genomic basis for their success in the environment. Here we present genomes from 16 axenic cultures of acI Actinobacteria. The isolates were not only of minute cell size, but also among the most streamlined free-living microbes, with extremely small genome sizes (1.2-1.4 Mbp) and low genomic GC content. Genome reduction in these bacteria might have led to auxotrophy for various vitamins, amino acids and reduced sulphur sources, thus creating dependencies to co-occurring organisms (the 'Black Queen' hypothesis). Genome analyses, moreover, revealed a surprising degree of inter- and intraspecific diversity in metabolic pathways, especially of carbohydrate transport and metabolism, and mainly encoded in genomic islands. The striking genotype microdiversification of acI Actinobacteria might explain their global success in highly dynamic freshwater environments with complex seasonal patterns of allochthonous and autochthonous carbon sources. We propose a new order within Actinobacteria ('Candidatus Nanopelagicales') with two new genera ('Candidatus Nanopelagicus' and 'Candidatus Planktophila') and nine new species.


Assuntos
Actinobacteria/genética , Água Doce/microbiologia , Genoma Bacteriano , Actinobacteria/classificação , Actinobacteria/isolamento & purificação , Composição de Bases , Biodiversidade , DNA Bacteriano/química , Redes e Vias Metabólicas/genética , Filogenia
6.
Sci Rep ; 7(1): 13770, 2017 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-29062037

RESUMO

After strong fertilization in the 20th century, many deep lakes in Central Europe are again nutrient poor due to long-lasting restoration (re-oligotrophication). In line with reduced phosphorus and nitrogen loadings, total organismic productivity decreased and lakes have now historically low nutrient and biomass concentrations. This caused speculations that restoration was overdone and intended fertilizations are needed to ensure ecological functionality. Here we show that recent re-oligotrophication processes indeed accelerated, however caused by lake warming. Rising air temperatures strengthen thermal stabilization of water columns which prevents thorough turnover (holomixis). Reduced mixis impedes down-welling of oxygen rich epilimnetic (surface) and up-welling of phosphorus and nitrogen rich hypolimnetic (deep) water. However, nutrient inputs are essential for algal spring blooms acting as boost for annual food web successions. We show that repeated lack (since 1977) and complete stop (since 2013) of holomixis caused drastic epilimnetic phosphorus depletions and an absence of phytoplankton spring blooms in Lake Zurich (Switzerland). By simulating holomixis in experiments, we could induce significant vernal algal blooms, confirming that there would be sufficient hypolimnetic phosphorus which presently accumulates due to reduced export. Thus, intended fertilizations are highly questionable, as hypolimnetic nutrients will become available during future natural or artificial turnovers.


Assuntos
Biomassa , Eutrofização/fisiologia , Lagos/química , Estações do Ano , Movimentos da Água , Monitoramento Ambiental , Nitrogênio/análise , Fósforo/análise
7.
J Dairy Sci ; 100(1): 402-411, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27865493

RESUMO

Supernumerary teats (SNT) are any abnormal teats found on a calf in addition to the usual and functional 4 teats. The presence of SNT has also been termed "hyperthelia" since the end of the 19th century. Supernumerary teats can act as an incubator for bacteria, infecting the whole udder, and can interfere with the positioning of the milking machine, and consequently, have economic relevance. Different types of SNT are observed at different positions on the udder. Caudal teats are in the rear, ramal teats are attached to another teat, and intercalary teats are found between 2 regular teats. Not all teats are equally developed; some are completely functional but most are rudimentary and not attached to any mammary gland tissue. Recently, different studies showed the poly/oligogenic character of these malformations in cattle as well as in other mammalian species. The objective of this study was to analyze the genetic architecture and incidence of hyperthelia in Swiss Brown Swiss cattle using both traditional genetic evaluation as well as imputed whole genome sequence variant information. First, phenotypes collected over the last 20 yr were used together with pedigree information for estimation of genetic variance. Second, breeding values of Brown Swiss bulls were estimated applying the BLUP algorithm. The BLUP-EBV were deregressed and used as phenotypes in genome-wide association studies. The gene LGR5 on chromosome 5 was identified as a candidate for the presence of SNT. Using alternative trait coding, genomic regions on chromosome 17 and 20 were also identified as being involved in the development of SNT with their own supernumerary mammary gland tissue. Implementing knowledge gained in this study as a routine application allows a more accurate evaluation of the trait and reduction of SNT prevalence in the Swiss Brown Swiss cattle population.


Assuntos
Estudo de Associação Genômica Ampla/veterinária , Genoma , Animais , Cruzamento , Bovinos , Feminino , Genômica , Glândulas Mamárias Animais , Mamilos
8.
ISME J ; 9(11): 2442-53, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25942006

RESUMO

Methylotrophic planktonic bacteria fulfill a particular role in the carbon cycle of lakes via the turnover of single-carbon compounds. We studied two planktonic freshwater lineages (LD28 and PRD01a001B) affiliated with Methylophilaceae (Betaproteobacteria) in Lake Zurich, Switzerland, by a combination of molecular and cultivation-based approaches. Their spatio-temporal distribution was monitored at high resolution (n=992 samples) for 4 consecutive years. LD28 methylotrophs constituted up to 11 × 10(7) cells l(-1) with pronounced peaks in spring and autumn-winter, concomitant with blooms of primary producers. They were rare in the warm water layers during summer but abundant in the cold hypolimnion, hinting at psychrophilic growth. Members of the PRD01a001B lineage were generally less abundant but also had maxima in spring. More than 120 axenic strains from these so far uncultivated lineages were isolated from the pelagic zone by dilution to extinction. Phylogenetic analysis separated isolates into two distinct genotypes. Isolates grew slowly (µmax=0.4 d(-1)), were of conspicuously small size, and were indeed psychrophilic, with higher growth yield at low temperatures. Growth was enhanced upon addition of methanol and methylamine to sterile lake water. Genomic analyses of two strains confirmed a methylotrophic lifestyle with a reduced set of genes involved in C1 metabolism. The very small and streamlined genomes (1.36 and 1.75 Mb) shared several pathways with the marine OM43 lineage. As the closest described taxa (Methylotenera sp.) are only distantly related to either set of isolates, we propose a new genus with two species, that is, 'Candidatus Methylopumilus planktonicus' (LD28) and 'Candidatus Methylopumilus turicensis' (PRD01a001B).


Assuntos
Betaproteobacteria/genética , Ciclo do Carbono , Água Doce/microbiologia , Methylophilaceae/genética , Temperatura Baixa , DNA Bacteriano/genética , DNA Ribossômico/genética , Ecologia , Água Doce/química , Genoma Bacteriano , Genótipo , Lagos/microbiologia , Funções Verossimilhança , Metanol/química , Methylophilaceae/classificação , Filogenia , Plâncton/genética , RNA Ribossômico 16S/genética , Reprodutibilidade dos Testes , Estações do Ano , Análise de Sequência de DNA , Suíça , Microbiologia da Água
9.
Front Microbiol ; 6: 247, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25873914

RESUMO

Flow cytometric sorting is a powerful tool to physically separate cells within mixed microbial communities. If combined with phylogenetic staining (fluorescence in situ hybridization, FISH) it allows to specifically sort defined genotypic microbial populations from complex natural samples. However, the targeted enrichment of freshwater ultramicrobacteria, such as members of the LD12 clade of Alphaproteobacteria (SAR11-IIIb), is still challenging. Current FISH protocols, even in combination with signal amplification by catalyzed reporter deposition (CARD), are not sufficiently sensitive for the distinction of these bacteria from background noise by flow cytometry, presumably due to their low ribosome content and small cell sizes. We, therefore, modified a CARD based flow sorting protocol with the aim of increasing its sensitivity to a level sufficient for ultramicrobacteria. This was achieved by a second signal amplification step mediated by horseradish peroxidase labeled antibodies targeted to the fluorophores that were previously deposited by CARD-FISH staining. The protocol was tested on samples from an oligo-mesotrophic lake. Ultramicrobacteria affiliated with LD12 Alphaproteobacteria could be successfully sorted to high purity by flow cytometry. The ratios of median fluorescence signal to background ranged around 20, and hybridization rates determined by flow cytometry were comparable to those obtained by fluorescence microscopy. Potential downstream applications of our modified cell staining approach range from the analysis of microdiversity within 16S rRNA-defined populations to that of functional properties, such as the taxon-specific incorporation rates of organic substrates.

10.
Environ Microbiol ; 17(3): 781-95, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24903166

RESUMO

We studied the seasonal growth potential of opportunistic bacterial populations in Lake Zurich (Switzerland) by a series of grazer-free dilution culture assays. Pronounced shifts in the composition of the bacterial assemblages were observed within one doubling of total cell numbers, from initially abundant Actinobacteria to other fast-growing microbial lineages. Small populations with growth potentials far above community average were detected throughout the year with striking seasonal differences in their respective taxonomic affiliations. Members of Cytophaga-Flavobacteria (CF) were disproportionally proliferating only during phytoplankton blooms in spring and summer, while Beta- and Gammaproteobacteria showed superior growth at all other occasions. Growth rates of Alphaproteobacteria and esp. Sphingomonadaceae were significantly correlated to water temperatures and were far above community average in summer. Within the genus Flavobacterium, two species-like populations showed a tendency for fast growth in most experiments, while four others were exclusively proliferating either during a spring or during a summer phytoplankton bloom. Their high growth potentials but low in situ abundances hint at a tight control by bacterivorous grazers and at a consequently accelerated carbon flux to higher trophic levels.


Assuntos
Bactérias/classificação , Bactérias/crescimento & desenvolvimento , Ciclo do Carbono , Lagos/microbiologia , Fitoplâncton/crescimento & desenvolvimento , Actinobacteria/crescimento & desenvolvimento , Alphaproteobacteria/crescimento & desenvolvimento , Betaproteobacteria/crescimento & desenvolvimento , Carbono/metabolismo , Cytophaga/crescimento & desenvolvimento , Ecossistema , Flavobacteriaceae/crescimento & desenvolvimento , Estações do Ano , Suíça , Temperatura
11.
Phys Rev Lett ; 109(5): 057402, 2012 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-23006206

RESUMO

Direct-gap gain up to 850 cm(-1) at 0.74 eV is measured and modeled in optically pumped Ge-on-Si layers for photoexcited carrier densities of 2.0 × 10(20) cm(-3). The gain spectra are correlated to carrier density via plasma-frequency determinations from reflection spectra. Despite significant gain, optical amplification cannot take place, because the carriers also generate pump-induced absorption of ≈7000 cm(-1). Parallel studies of III-V direct-gap InGaAs layers validate our spectroscopy and modeling. Our self-consistent results contradict current explanations of lasing in Ge-on-Si cavities.

12.
Mol Cell Probes ; 26(6): 248-52, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22405934

RESUMO

In the Swiss Large White pig population a genetically caused arthrogryposis multiplex congenita (AMC) variant was identified. The disease is autosomal recessively inherited and is a fatal defect. Affected piglets are of normal size, but show malformed and permanently contracted joints in their legs. Often the spinal cord is curved and the lower jaw is shortened. Originally, AMC was mapped to a 5 Mb region on pig chromosome 5 (SSC5) between microsatellite markers SW152 and SW904. In order to detect unaffected carriers a diagnostic test using markers within the candidate region was developed. However, two independent recombination events occurred in a diseased and in a healthy piglet. Therefore, we selected 24 consecutive markers (3 microsatellites, 19 SNPs and 2 indels) in the candidate region, and determined the haplotypes in the two pedigrees with the recombinations. The parents and five offspring were investigated. In consequence, we were able to narrow down the candidate region and map AMC between SNPs ALGA0032767 and DRGA0006010 on SSC5 which span around 2.32 Mb. The candidate region shares homology to human chromosome 12. However, we are still lacking good candidate genes. A PCR-RFLP was developed and is used as an improved genetic test for AMC.


Assuntos
Artrogripose/veterinária , Estudos de Associação Genética , Doenças dos Suínos/diagnóstico , Animais , Artrogripose/diagnóstico , Artrogripose/genética , Feminino , Haplótipos , Desequilíbrio de Ligação , Masculino , Técnicas de Diagnóstico Molecular , Linhagem , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Sus scrofa , Suínos , Doenças dos Suínos/genética
13.
BMC Res Notes ; 4: 225, 2011 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-21718470

RESUMO

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) that express the F4ab and F4ac fimbriae is a major contributor to diarrhoea outbreaks in the pig breeding industry, infecting both newborn and weaned piglets. Some pigs are resistant to this infection, and susceptibility is inherited as a simple dominant Mendelian trait. Indentifying the genetics behind this trait will greatly benefit pig welfare as well as the pig breeding industry by providing an opportunity to select against genetically susceptible animals, thereby reducing the number of diarrhoea outbreaks. The trait has recently been mapped by haplotype sharing to a 2.5 Mb region on pig chromosome 13, a region containing 18 annotated genes. FINDINGS: The coding regions of five candidate genes for susceptibility to ETEC F4ab/ac infection (TFRC, ACK1, MUC20, MUC4 and KIAA0226), all located in the 2.5 Mb region, were investigated for the presence of possible causative mutations. A total of 34 polymorphisms were identified in either coding regions or their flanking introns. The genotyping data for two of those were found to perfectly match the genotypes at the ETEC F4ab/ac locus, a G to C polymorphism in intron 11 of TFRC and a C to T silent polymorphism in exon 22 of KIAA0226. Transcriptional profiles of the five genes were investigated in a porcine tissue panel including various intestinal tissues. All five genes were expressed in intestinal tissues at different levels but none of the genes were found differentially expressed between ETEC F4ab/ac resistant and ETEC F4ab/ac susceptible animals in any of the tested tissues. CONCLUSIONS: None of the identified polymorphisms are obvious causative mutations for ETEC F4ab/ac susceptibility, as they have no impact on the level of the overall mRNA expression nor predicted to influence the composition of the amino acids composition. However, we cannot exclude that the five tested genes are bona fide candidate genes for susceptibility to ETEC F4ab/ac infection since the identified polymorphism might affect the translational apparatus, alternative splice forms may exist and post translational mechanisms might contribute to disease susceptibility.

14.
Blood ; 117(23): 6227-36, 2011 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-21460242

RESUMO

Gastric marginal zone B-cell lymphoma of MALT type (MALT lymphoma) arises in the context of chronic inflammation induced by the bacterial pathogen Helicobacter pylori. Although generally considered an indolent disease, MALT lymphoma may transform to gastric diffuse large B-cell lymphoma (gDLBCL) through mechanisms that remain poorly understood. By comparing microRNA expression profiles of gastric MALT lymphoma and gDLBCL, we have identified a signature of 27 deregulated microRNAs(miRNAs) that share the characteristic of being transcriptionally repressed by Myc. Myc overexpression was consequently detected in 80% of gDLBCL but only 20% of MALT lymphomas spotted on a tissue microarray. A highly similar signature of Myc-repressed miRNAs was further detected in nodal DLBCL. Small interfering RNA-mediated knock-down of Myc blocked proliferation of DLBCL cell lines. Of the Myc-repressed miRNAs down-regulated in malignant lymphoma, miR-34a showed the strongest antiproliferative properties when overexpressed in DLBCL cells. We could further attribute miR-34a's tumor-suppressive effects to deregulation of its target FoxP1. FoxP1 overexpression was detected in gDLBCL but not in gastric MALT lymphoma; FoxP1 knock-down efficiently blocked DLBCL proliferation. In conclusion, our results elucidate a novel Myc- and FoxP1-dependent pathway of malignant transformation and suggest miR-34a replacement therapy as a promising strategy in lymphoma treatment.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Linfoma de Zona Marginal Tipo Células B/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , MicroRNAs/metabolismo , RNA Neoplásico/metabolismo , Proteínas Repressoras/metabolismo , Neoplasias Gástricas/metabolismo , Linhagem Celular Tumoral , Fatores de Transcrição Forkhead/genética , Infecções por Helicobacter/genética , Infecções por Helicobacter/metabolismo , Helicobacter pylori , Humanos , Linfoma de Zona Marginal Tipo Células B/genética , Linfoma Difuso de Grandes Células B/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , Proteínas Repressoras/genética , Neoplasias Gástricas/genética
15.
Eur J Hum Genet ; 19(5): 555-60, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21326286

RESUMO

In this study, we report a familial inversion of chromosome 18, inv(18)(p11.31q21.33), in both members of a consanguineous couple. Their first child had inherited one balanced pericentric inversion along with a recombinant chromosome 18 resulting in dup(18q)/del(18p), and had mild dysmorphic features in the absence of mental and developmental retardation. The second child had received two recombinant chromosomes 18, from the mother a derivative chromosome 18 with dup(18p)/del(18q) and from the father a derivative chromosome 18 with dup(18q)/del(18p). The aberration was prenatally detected; however, as the two opposite aneuploidies were thought to compensate each other, the family decided to carry on with the pregnancy, knowing that uniparental disomy for the segments outside the inversion could have an adverse influence on the development of the child. Uniparental disomy was confirmed by SNP arrays. The child, who has been followed up until the age of 20 months, is healthy and normal. It seems to be the first reported case with two opposite recombinant chromosomes that compensate each other and lead to segmental uniparental disomy for two segments on the chromosome, one maternal and the other paternal.


Assuntos
Inversão Cromossômica , Cromossomos Humanos Par 18 , Dissomia Uniparental , Criança , Pré-Escolar , Consanguinidade , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Linhagem
16.
Can J Microbiol ; 57(2): 105-14, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21326352

RESUMO

Glacier forefield environments are exposed to extreme and fluctuating climatic and nutritional conditions. The high diversity of free-living diazotrophic communities found in these environments indicates that nitrogen fixers are able to efficiently cope with such conditions. In this study, a nifH microarray was used to monitor changes in diazotrophic populations in the field over a season, in the presence or absence of plants and in 2 glacier forefields characterized by a different bedrock type (siliceous or calcareous), as well as at different temperatures (10 °C, 15 °C) and under different nitrogen fertilization regimes (0, 10, 40 kg N·ha(-1)·year(-1)) in laboratory systems. Population structures responded highly dynamically to environmental changes. Plant presence had the strongest impact, which decreased toward the end of the season and with high amounts of nitrogen fertilization. Temperature and nitrogen fertilization increases indirectly affected diazotrophic communities through their positive impact on plant growth. These results indicate strong carbon limitation in young glacier forefield soils. Phylotypes related to the genus Methylocystis strongly responded to environmental variations. These methanotrophic microorganisms, which are able to retrieve nitrogen and carbon from the atmospheric pool, are particularly adapted to the extreme nutritional conditions found in glacier forefields.


Assuntos
Monitoramento Ambiental/métodos , Camada de Gelo/microbiologia , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredutases/análise , Microbiologia do Solo , Carbono/química , Nitrogênio/química , Fixação de Nitrogênio , Desenvolvimento Vegetal , Rizosfera , Solo/análise , Temperatura
17.
Mamm Genome ; 22(1-2): 122-9, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21136063

RESUMO

Enterotoxigenic Escherichia coli (ETEC) with fimbriae of the F4 family are one of the major causes of diarrhea and death among neonatal and young piglets. Bacteria use the F4 fimbriae to adhere to specific receptors expressed on the surface of the enterocytes. F4 fimbriae exist in three different antigenic variants, F4ab, F4ac, and F4ad, of which F4ac is the most common. Resistance to ETEC F4ab/F4ac adhesion in pigs has been shown to be inherited as an autosomal recessive trait. In previous studies the ETEC F4ab/F4ac receptor locus (F4bcR) was mapped to the q41 region on pig chromosome 13. A polymorphism within an intron of the mucin 4 (MUC4) gene, which is one of the possible candidate genes located in this region, was shown earlier to cosegregate with the F4bcR alleles. Recently, we discovered a Large White boar from a Swiss experimental herd with a recombination between F4bcR and MUC4. A three-generation pedigree including 45 offspring was generated with the aim to use this recombination event to refine the localization of the F4bcR locus. All pigs were phenotyped using the microscopic adhesion test and genotyped for a total of 59 markers. The recombination event was mapped to a 220-kb region between a newly detected SNP in the leishmanolysin-like gene (LMLN g.15920) and SNP ALGA0072075. In this study the six SNPs ALGA0072075, ALGA0106330, MUC13-226, MUC13-813, DIA0000584, and MARC0006918 were in complete linkage disequilibrium with F4bcR. Based on this finding and earlier investigations, we suggest that the locus for F4bcR is located between the LMLN locus and microsatellite S0283.


Assuntos
Escherichia coli Enterotoxigênica/imunologia , Infecções por Escherichia coli/veterinária , Fímbrias Bacterianas/genética , Mucina-4/genética , Receptores de Superfície Celular/genética , Doenças dos Suínos/genética , Suínos/genética , Animais , Aderência Bacteriana , Sequência de Bases , Mapeamento Cromossômico , Escherichia coli Enterotoxigênica/genética , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Feminino , Fímbrias Bacterianas/imunologia , Desequilíbrio de Ligação , Masculino , Dados de Sequência Molecular , Mucina-4/imunologia , Polimorfismo de Nucleotídeo Único , Receptores de Superfície Celular/imunologia , Suínos/imunologia , Suínos/microbiologia , Doenças dos Suínos/imunologia , Doenças dos Suínos/microbiologia
18.
Eur J Hum Genet ; 18(12): 1315-21, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20648054

RESUMO

Aortic dilatation/dissection (AD) can occur spontaneously or in association with genetic syndromes, such as Marfan syndrome (MFS; caused by FBN1 mutations), MFS type 2 and Loeys-Dietz syndrome (associated with TGFBR1/TGFBR2 mutations), and Ehlers-Danlos syndrome (EDS) vascular type (caused by COL3A1 mutations). Although mutations in FBN1 and TGFBR1/TGFBR2 account for the majority of AD cases referred to us for molecular genetic testing, we have obtained negative results for these genes in a large cohort of AD patients, suggesting the involvement of additional genes or acquired factors. In this study we assessed the effect of COL3A1 deletions/duplications in this cohort. Multiplex ligation-dependent probe amplification (MLPA) analysis of 100 unrelated patients identified one hemizygous deletion of the entire COL3A1 gene. Subsequent microarray analyses and sequencing of breakpoints revealed the deletion size of 3,408,306 bp at 2q32.1q32.3. This deletion affects not only COL3A1 but also 21 other known genes (GULP1, DIRC1, COL5A2, WDR75, SLC40A1, ASNSD1, ANKAR, OSGEPL1, ORMDL1, LOC100129592, PMS1, MSTN, C2orf88, HIBCH, INPP1, MFSD6, TMEM194B, NAB1, GLS, STAT1, and STAT4), mutations in three of which (COL5A2, SLC40A1, and MSTN) have also been associated with an autosomal dominant disorder (EDS classical type, hemochromatosis type 4, and muscle hypertrophy). Physical and laboratory examinations revealed that true haploinsufficiency of COL3A1, COL5A2, and MSTN, but not that of SLC40A1, leads to a clinical phenotype. Our data not only emphasize the impact/role of COL3A1 in AD patients but also extend the molecular etiology of several disorders by providing hitherto unreported evidence for true haploinsufficiency of the underlying gene.


Assuntos
Doenças da Aorta/genética , Colágeno Tipo III/genética , Colágeno Tipo V/genética , Haploinsuficiência/genética , Hemizigoto , Miostatina/genética , Deleção de Sequência/genética , Doenças da Aorta/patologia , Pareamento de Bases/genética , Sequência de Bases , Quebra Cromossômica , Colágeno Tipo III/ultraestrutura , Colágeno Tipo V/ultraestrutura , Sondas de DNA/metabolismo , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Fenótipo , Reação em Cadeia da Polimerase
19.
Environ Microbiol ; 11(8): 2179-89, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19453699

RESUMO

Functional microarrays are powerful tools that allow the parallel detection of multiple strains at the species level and therefore to rapidly obtain information on microbial communities in the environment. However, the design of suitable probes is prone to uncertainties, as it is based so far on in silico predictions including weighted mismatch number and Gibbs free-energy values. This study describes the experimental selection of probes targeting subsequences of the nifH gene to study the community structure of diazotrophic populations present in Damma glacier (Swiss Central Alps) forefield soils. Using the Geniom One in situ synthesis technology (Febit, Germany), 2727 in silico designed candidate probes were tested. A total of 946 specific probes were selected and validated. This probe set covered a large diversity of the NifH phylotypes (35 out of the 45) found in the forefield. Hybridization predictors were tested statistically. Gibbs free-energy value for probe-target binding gave the best prediction for hybridization efficiency, while the weighted mismatch number was not significantly associated to probe specificity. In this study, we demonstrate that extensive experimental tests of probe-hybridization behaviour against sequences present in the studied environment remain a prerequisite for meaningful probe selection.


Assuntos
Camada de Gelo/microbiologia , Fixação de Nitrogênio/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oxirredutases/genética , Simulação por Computador , Sondas de Oligonucleotídeos , Oxirredutases/análise , Solo/análise , Microbiologia do Solo
20.
Circulation ; 114(1 Suppl): I125-31, 2006 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-16820561

RESUMO

BACKGROUND: Heart valve tissue engineering is a promising strategy to overcome the lack of autologous growing replacements, particularly for the repair of congenital malformations. Here, we present a novel concept using human prenatal progenitor cells as new and exclusive cell source to generate autologous implants ready for use at birth. METHODS AND RESULTS: Human fetal mesenchymal progenitors were isolated from routinely sampled prenatal chorionic villus specimens and expanded in vitro. A portion was cryopreserved. After phenotyping and genotyping, cells were seeded onto synthetic biodegradable leaflet scaffolds (n=12) and conditioned in a bioreactor. After 21 days, leaflets were endothelialized with umbilical cord blood-derived endothelial progenitor cells and conditioned for additional 7 days. Resulting tissues were analyzed by histology, immunohistochemistry, biochemistry (amounts of extracellular matrix, DNA), mechanical testing, and scanning electron microscopy (SEM) and were compared with native neonatal heart valve leaflets. Fresh and cryopreserved cells showed comparable myofibroblast-like phenotypes. Genotyping confirmed their fetal origin. Neo-tissues exhibited organization, cell phenotypes, extracellular matrix production, and DNA content comparable to their native counterparts. Leaflet surfaces were covered with functional endothelia. SEM showed cellular distribution throughout the polymer and smooth surfaces. Mechanical profiles approximated those of native heart valves. CONCLUSIONS: Prenatal fetal progenitors obtained from routine chorionic villus sampling were successfully used as an exclusive, new cell source for the engineering of living heart valve leaflets. This concept may enable autologous replacements with growth potential ready for use at birth. Combined with the use of cell banking technology, this approach may be applied also for postnatal applications.


Assuntos
Bioprótese , Amostra da Vilosidade Coriônica , Vilosidades Coriônicas , Próteses Valvulares Cardíacas , Células-Tronco Mesenquimais/citologia , Engenharia Tecidual/métodos , Coleta de Tecidos e Órgãos , Implantes Absorvíveis , Biodegradação Ambiental , Reatores Biológicos , Separação Celular , Técnicas de Cocultura , Criopreservação , DNA/metabolismo , Células Endoteliais/citologia , Matriz Extracelular/metabolismo , Sangue Fetal/citologia , Citometria de Fluxo , Genótipo , Idade Gestacional , Humanos , Doadores Vivos , Masculino , Teste de Materiais , Células-Tronco Mesenquimais/metabolismo , Microscopia Eletrônica de Varredura , Técnicas de Cultura de Órgãos/métodos , Fenótipo , Resistência à Tração , Engenharia Tecidual/instrumentação , Preservação de Tecido , Transplante Autólogo
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